Ether-functionalized ionic liquids (ILs) usually have low viscosities, and can be designed to be compatible with enzymes. However, there is a lack of understanding of the effect of different ether-functionalized structures on the enzyme activity. We systematically evaluated new ether-functionalized ILs carrying different cation cores (pairing with Tf2N- anions) in two Novozym 435-catalyzed reactions: (1) the transesterification of ethyl sorbate with 1-propanol at 50 degrees C; (2) the ring-opening polymerization (ROP) of epsilon-caprolactone at 70 degrees C. The lipase showed different activities: in the first reaction, [CH3OCH2CH2-Et3N] [Tf2N] and [CH3OCH2CH2-Py] [Tf2N] gave the highest reaction rates; in the second reaction, [CH3OCH2CH2-PBu3] [Tf2N] produced the highest molecular mass (M-w, up to 25,400 Da). The lipase's thermal stability in [CH3OCH2CH2-Et3N] [Tf2N] was found much higher than that in t-butanol. The fluorescence spectra of free lipase (excited at 280 nm) in these ILs reveal that the wavelength of the maximum emission peak occurred at 314 nm for both [CH3OCH2CH2PBu3] [Tf2N] and [CH3OCH2CH2PEt3] [Tf2N], which matched closely with that (313 nm) in aqueous phosphate buffer (pH 7.5, 20 mM), while other ether-functionalized ILs led to various degrees of red shifts. In summary, the lipase activity is not only dependent on the IL structure, but also on the substrate and other reaction conditions.